Recombinant Schizosaccharomyces pombe Putative uncharacterized protein C806.11 (SPAC806.11)

What is currently known about putative uncharacterized protein C806.11 (SPAC806.11) in S. pombe?

SPAC806.11 belongs to a family of putative uncharacterized proteins in fission yeast. While specific literature on this protein is limited, related proteins in the SPAC806 region have been characterized. For instance, SPAC806.04c (renamed Duf8901) has been identified as a paralogous cobalt/nickel-dependent phosphatase/pyrophosphatase enzyme . Research approaches used for characterizing related proteins can provide methodological frameworks for studying SPAC806.11.

To begin characterization, researchers should consider sequence analysis to identify conserved domains, phylogenetic analysis to determine evolutionary relationships with characterized proteins, and preliminary expression analysis using RNA-seq data. These approaches provide foundational information before designing wet-lab experiments to determine protein function.

What experimental systems are appropriate for expressing recombinant SPAC806.11?

When expressing recombinant SPAC806.11, several expression systems can be considered, each with distinct advantages:

For initial characterization, an E. coli system with appropriate tags (His, GST) often provides sufficient protein for biochemical analysis. If protein function depends on post-translational modifications, native expression in S. pombe with appropriate tagging is recommended for maintaining physiological relevance.

How can I confirm the subcellular localization of SPAC806.11?

Determining subcellular localization provides crucial insights into protein function. A systematic approach includes:

• Bioinformatic prediction using tools like PSORT, TargetP, and TMHMM to identify potential localization signals.

• Fluorescent tagging: Generate C- or N-terminal GFP fusion constructs. Since S. pombe transcription relies on upstream activation sequence elements, ensure your construct design maintains proper regulatory sequences .

• Co-localization studies with established organelle markers to confirm bioinformatic predictions.

• Subcellular fractionation followed by Western blotting for biochemical verification of localization predictions.

• Immunogold electron microscopy for high-resolution localization if antibodies are available or can be developed.

When designing GFP fusion constructs, consider that S. pombe transcription initiates within approximately 25-40 base pairs downstream from the TATA element , which might influence expression efficiency of your constructs.

How should I design experiments to determine if SPAC806.11 has enzymatic activity similar to its paralog SPAC806.04c (Duf8901)?

Based on the paralogous relationship with SPAC806.04c (Duf8901), which functions as a cobalt/nickel-dependent phosphatase/pyrophosphatase , a systematic experimental approach would include:

• Sequence alignment analysis to identify conserved catalytic residues between SPAC806.11 and Duf8901.

• Recombinant protein expression and purification with appropriate metal cofactors (cobalt, nickel).

• In vitro enzymatic assays testing various substrates including:

  • pNPP (para-nitrophenyl phosphate) for phosphatase activity

  • Various pyrophosphate derivatives

  • Inositol pyrophosphates, given Duf8901's involvement in phosphate homeostasis

• Metal dependency assays to determine if activity requires cobalt/nickel or other divalent cations.

• Mutagenesis of predicted catalytic residues to confirm their involvement in any detected activity.

If enzymatic activity is detected, perform kinetic analysis (determining Km, Vmax, kcat) for quantitative characterization. Compare these parameters with those of Duf8901 to establish functional similarities or differences.

What experimental design would best determine if SPAC806.11 participates in transcriptional regulation like other proteins in S. pombe?

To investigate potential transcriptional regulatory functions of SPAC806.11, implement a multi-faceted experimental design:

• Generate a SPAC806.11 deletion strain using homologous recombination.

• Perform RNA-seq comparing wild-type and SPAC806.11Δ strains under various conditions (standard growth, nutrient limitation, stress).

• Identify differentially expressed genes and analyze them for common regulatory elements or pathways.

• Conduct chromatin immunoprecipitation followed by sequencing (ChIP-seq) using tagged SPAC806.11 to identify potential binding sites on DNA.

• Perform RNA polymerase II (Pol2) occupancy analysis to determine if SPAC806.11 affects transcription initiation or elongation.

Consider that S. pombe transcription initiation occurs within a narrow window approximately 25-40 base pairs downstream from the TATA element . If SPAC806.11 functions in transcriptional regulation, changes in start site selection might be observed in the deletion strain.

How can I design experiments to investigate if SPAC806.11 interacts with inositol pyrophosphates pathway components?

Given that related proteins interact with the inositol pyrophosphate (IP8) signaling pathway , a comprehensive interaction study would include:

• Co-immunoprecipitation (Co-IP) experiments with tagged SPAC806.11 and known IP8 pathway components (Asp1, Aps1, Spx1).

• Yeast two-hybrid screening to identify novel protein interactors.

• Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in vivo.

• Genetic interaction studies:

  • Generate double mutants of SPAC806.11Δ with mutations in IP8 pathway genes (asp1Δ, asp1-D333A, aps1Δ)

  • Assess phenotypes including growth, phosphate homeostasis, and transcriptional effects

  • Test if SPAC806.11Δ effects are dependent on IP8 synthesis by Asp1 kinase, similar to observations with Duf8901

• Assess if SPAC806.11 contains an SPX domain (common in proteins that bind inositol pyrophosphates) through structural prediction and validation experiments.

How should I analyze proteomic data to identify post-translational modifications of SPAC806.11?

Comprehensive post-translational modification (PTM) analysis requires sophisticated mass spectrometry approaches:

• Sample preparation:

  • Purify tagged SPAC806.11 from S. pombe using appropriate affinity methods

  • Perform parallel enrichment for specific PTMs (phosphorylation, glycosylation)

  • Digest with multiple proteases (not just trypsin) to improve sequence coverage

• Mass spectrometry analysis:

  • Use stable isotope dimethyl labeling for quantitative comparison between conditions

  • Employ 2D liquid chromatography separations to increase detection sensitivity

  • Implement electron transfer dissociation (ETD) fragmentation, which preserves labile modifications

• Data analysis:

  • Use multiple search algorithms to cross-validate results

  • Employ site localization algorithms to precisely identify modified residues

  • Calculate site occupancy percentages for each modification

• Validation experiments:

  • Generate site-specific mutants (e.g., S→A for phosphorylation sites)

  • Assess functional consequences through phenotypic analysis

  • Perform targeted quantification of key modifications using parallel reaction monitoring

For glycosylation analysis specifically, consider lectin affinity chromatography enrichment methods. Concanavalin A (Con A) exhibits high affinity to high-mannose type N-glycans, while wheat germ agglutinin (WGA) is selective for N-acetyl-glucosamine (GlcNAc) .

How do I reconcile conflicting experimental results about SPAC806.11 function?

When facing contradictory experimental results, apply a systematic troubleshooting approach:

• Assess experimental design variables using design of experiments (DOE) principles:

  • Identify independent variables that might influence results (strain background, growth conditions, purification methods)

  • Determine if control variables were properly maintained across experiments

  • Evaluate statistical power of each experiment to determine if sample sizes were sufficient

• Consider biological explanations for apparent contradictions:

  • Multifunctional proteins can show context-dependent activities

  • Genetic background effects might explain strain-specific results

  • Compensatory mechanisms might mask phenotypes in deletion studies

• Apply critical thinking to experimental evidence:

  • Evaluate methodological strengths and limitations of each conflicting result

  • Consider if differences in protein tags or expression levels explain contradictions

  • Assess if different experimental timepoints could explain temporal differences in results

• Design reconciliation experiments:

  • Create controlled experiments that specifically test conditions where contradictions appeared

  • Use orthogonal methods to validate key findings

  • Consider conditional or partial loss-of-function approaches rather than complete deletions

Document all variables between conflicting experiments in a comprehensive table, which often reveals overlooked differences in experimental conditions that explain apparent contradictions.

What are the optimal protein extraction and purification methods for SPAC806.11 biochemical characterization?

Effective extraction and purification of SPAC806.11 requires optimization of several parameters:

For membrane-associated proteins, consider using digitonin or DDM as gentler detergents. If SPAC806.11 shares characteristics with Duf8901, include divalent cations (Co2+ or Ni2+) in buffers to stabilize protein structure and maintain enzymatic activity .

Include an experimental control by performing parallel purification from a strain lacking the tagged protein to identify non-specific contaminants in your purification.

How can I develop a reliable assay to measure SPAC806.11 activity in vitro?

Developing a robust activity assay requires careful consideration of protein function and detection methods:

• If SPAC806.11 potentially functions as a phosphatase/pyrophosphatase like Duf8901 :

  • Primary screen: use colorimetric substrates like p-nitrophenyl phosphate

  • Secondary validation: use physiologically relevant substrates (inositol pyrophosphates)

  • Include proper controls: heat-inactivated enzyme, catalytic site mutants

• Assay optimization parameters:

  • Buffer composition: test multiple pH values (6.0-8.0) and salt concentrations

  • Metal dependency: screen divalent cations (Mg2+, Mn2+, Co2+, Ni2+, Zn2+)

  • Temperature and time dependence: establish linear range of activity

  • Substrate concentration: determine Km and Vmax

• Detection methods:

  • For phosphatase activity: malachite green assay for phosphate release

  • For more complex substrates: couple activity to fluorescent or luminescent detection systems

  • For high-throughput screening: adapt to microplate format with automated liquid handling

• Validation criteria:

  • Signal-to-noise ratio >10:1

  • Z-factor >0.7 for robust assays

  • Reproducibility across independent protein preparations (CV <15%)

What approaches can determine if SPAC806.11 functions in the phosphate homeostasis pathway?

To investigate involvement in phosphate homeostasis, implement a multi-level experimental approach:

• Phenotypic characterization:

  • Compare growth of wild-type and SPAC806.11Δ strains under phosphate limitation

  • Measure acid phosphatase activity as a reporter of PHO pathway activation

  • Quantify intracellular and secreted phosphate levels

• Genetic interaction studies:

  • Create double mutants with known phosphate homeostasis genes (pho1, pho84, pho7)

  • Generate SPAC806.11Δ asp1-D333A double mutants to test dependency on IP8 synthesis

  • Test epistatic relationships by measuring reporter gene expression

• Transcriptional analysis:

  • Measure expression of PHO regulon genes in SPAC806.11Δ using RT-qPCR

  • Analyze prt lncRNA transcription termination, which regulates pho1 expression

  • Assess if SPAC806.11 deletion affects Pol2 CTD phosphorylation patterns

• Biochemical approaches:

  • Test if SPAC806.11 interacts with transcription termination factors (Rhn1, CPF subunits)

  • Measure IP8 levels in SPAC806.11Δ compared to wild-type

  • Determine if SPAC806.11 contains domains that interact with inositol pyrophosphates

If SPAC806.11 functions in phosphate homeostasis, deletion effects might depend on the Asp1 kinase activity that synthesizes IP8, similar to what has been observed with other factors in this pathway .

What strategies can overcome challenges in expressing soluble recombinant SPAC806.11?

When facing solubility issues with recombinant SPAC806.11, implement a systematic optimization approach:

• Expression system modifications:

  • Test multiple expression vectors with different promoter strengths

  • Evaluate various host strains (BL21(DE3), Rosetta, SHuffle for E. coli)

  • Consider codon optimization for the expression host

  • Try fusion partners known to enhance solubility (MBP, SUMO, TrxA)

• Expression condition optimization:

  • Reduce induction temperature (16-20°C)

  • Lower inducer concentration

  • Extend expression time (overnight at lower temperatures)

  • Add chemical chaperones to media (glycerol, sorbitol, arginine)

• Protein engineering approaches:

  • Express individual domains rather than full-length protein

  • Remove predicted disordered regions

  • Introduce surface mutations to enhance solubility

  • Design constructs based on comparative analysis with soluble homologs

• Purification strategies for challenging proteins:

  • On-column refolding during purification

  • Inclusion of stabilizing ligands or cofactors in buffers

  • Detergent screening for membrane-associated proteins

  • Optimization of buffer components (salt concentration, pH, additives)

Maintain a systematic record of all conditions tested and resulting protein solubility/activity to identify patterns that might inform successful expression strategies.

How can I validate that phenotypes observed in SPAC806.11Δ strains are specifically due to loss of this protein?

To establish causality between SPAC806.11 deletion and observed phenotypes:

• Complementation testing:

  • Reintroduce wild-type SPAC806.11 on a plasmid or integrated into a neutral locus

  • Include proper controls with empty vector

  • Test multiple expression levels to avoid artifacts from overexpression

  • Use the native promoter when possible to maintain physiological expression patterns

• Generate and test point mutants:

  • Create catalytic site mutants if enzymatic activity is suspected

  • Mutate potential protein interaction surfaces

  • Test domain deletion constructs to identify functional regions

• Address potential off-target effects:

  • Sequence the deletion strain to confirm precise gene removal

  • Check expression of neighboring genes to rule out polar effects

  • Generate the deletion using multiple independent methods and compare phenotypes

• Consider genetic background effects:

  • Test phenotypes in multiple strain backgrounds

  • Create marker-free deletions to eliminate marker gene influence

  • Use heterozygous diploids to assess haploinsufficiency or dominant effects